The Determination of Total Dissolved Nitrogen in Streamwater

 

 

Modification of 4500-Norg D (Standard Methods for the Examination of Water and Wastewater- APHA 1995, 19^th edition).  This is a modification of the method used by Neil Bettez (also some modifications by S. Thomas and Adrian Green). The persulfate concentration and type of acid were changed.

 

 

This method measures the amount of total dissolved nitrogen (NH₄ ⁺, NO₃^-, and organic N) in a water sample.  The original method assumes that the sample is NOT acidified, however, modifications were made to the amount of NaOH added to account for sample acidification (ACG 1/03).  A 10mL sample is digested by autoclaving the sample and a persulfate solution.  The digestion causes all forms of N to be oxidized to NO₃^-.  The pH of the sample is then readjusted with acid and analyzed on the Alpkem or Lachat for NO₃^- (which is the concentration of TDN in the sample).  Care must be taken to ensure complete digestion.  Organic reference materials must be used to check that the persulfate digestion has gone to completion.  The standards and reference materials get digested in the exact same manner as the samples.

 

Method       

 

·       Measure 10mL of standard/sample into a clean, pre-calibrated, black-capped (teflon liner) digestion tube

·       Add 2mL K₂S₂O₈ solution (0.05g/mL—see note below!)

·       Add 0.23 mL of 5.6M NaOH*

·       Cap TIGHTLY and shake

·       Autoclave for 1 hour

·       Let cool and add 0.08 mL (80 ml) of 5.6N HCL** to each sample (if sample will be analyzed right away)

·       Transfer samples to glass scint vials and determine N concentration on autoanalyzer.

or

·       Leave samples in digestion tubes and analyze on the Lachat directly.

 

*original method calls for 0.21 ml NaOH. Since our samples were acidified in the field with 0.1ml of 6N HCL per 50 ml of sample, we adjusted the volume of NaOH in order to bring the pH to 6-7.

**original method calls for 0.4 ml of 0.9N H2SO4. We chose to use HCL, since that is what our samples were acidified with initially.

 

Reagents

 

5.6M NaOH

Add 224 g NaOH slowly to DI in a volumetric placed in an ice bath.  When all NaOH is dissolved and volumetric has returned to room temperature, qs to 1L.  Store NaOH in a plastic container.

 

K₂S₂O₈

Make only as much K₂S₂O₈ as you will need for one day, as it degrades over time. Add 5g K₂S₂O₈  per 100mL DI and stir until dissolved (with a little bit of heat; it will take some time to dissolve)

NOTE:  The K₂S₂O₈ used is low P and low N persulfate from JT Baker (VWR# JT3239-1).

 

0.9N H₂SO₄

Add 25 mL concentrated H₂SO₄  to DI and qs to 1L

NOTE:  Make fresh to avoid NH₄ ⁺ absorption (i.e., don't use that old stuff you made months ago).

 

5.6N HCL

Add 460 mL of 12.1 N HCL to DI and bring up to IL with DI.

 

 

Standards-

 

1,000µM N/L Stock

Weigh  0.101g dried KNO₃ and add to a 1L volumetric

dissolve completely in DI

bring up to 1L with DI

transfer to an acid-washed bottle, add a few drops of chloroform, and store in the refrigerator

 

 

Working Standards 

 

Bring the stock 1,000µM N/L stock solution to room temperature

 

100µM N/L working standard - Dilute 10 ml of 1000 µM N/L stock in 100 ml DI

 

Standards:

 

0µM-    DI only

 

1µM-  1mL of 100µM and dilute with DI to 100mL   (1*100/100 = 1)

         or, 0.1 ml of 1000µM brought to 100 mL with DI

 

2µM-   2mL of 100µM and dilute with DI to 100mL  (2*100/100 = 2)

         or, 0.2 mL ml of 1000µM brought to 100 mL with DI

 

5µM-   5mL of 100µM and dilute with DI to 100mL  (5*100/100 = 5)

         or, 0.5 mL ml of 1000µM brought to 100 mL with DI

 

10µM-   etc….

20µM-  

30µM- 

 

notes: Since our samples were acidified in the field, we acidified our standards to the same Normality as the samples.

 

We digested a standard curve along with the samples, and used the digested curve to calculate sample concentrations. (ACG)

 

Standard Reference Materials

Use one or more of the below organics to check that the persulfate is converting organic N to inorganic N.  It is best to make one reference standard at the highest level of standard you're using and then another that's about the average concentration of your samples (to check digestion efficiency at the far range and to check the standard curve in the range where most of the samples are).

 

Nicotinic Acid p-toluenesulfonate (C₁₃H₁₃O₅SN)

This is supposed to be a difficult organic standard to digest and is ideal for a digestion check standard.

Make a primary standard by dissolving 0.2953g nicotinic acid p-toluesulfonate in DI.  qs to 1L.

 

 Dilute to the proper standard and sample range.

 

Nitrophenol

Make a primary standard of 4,000µM (4mM) by dissolving 0.278g nitrophenol in 500mL DI.

 

Make a working 40µM standard by diluting 1mL of 4,000µM in 100 mL DI.

 

 

ATP (C₁₀H₁₄N₅O₁₃P₃Na₂)

(This is used as a SRM for TDP but may also be used for TDN as well.)

Make a primary 2,000µMP standard by dissolving 0.1835g ATP in 500mL

 

Make a working 33.3µMP standard by dissolving 1mL 2,000µM standard in 100mL

NOTE: This working standard also contains 33.3µMN

(NOTE: but ATP is 7% water, so solution is not 33.3µM, but actually 31µM)

 

QCSPEX

Fisher makes a QC standard that contains N and P forms that can be used to check the accuracy of the standards.  This reference material will NOT challenge the digestion and should not be used to check the digestion efficiency, but it is a helpful reference material to check the accuracy of your standards.

 

 

 

Supplies

 

Screw top test tubes             Fisher #14-930-10E

Additional teflon-lined caps    Fisher # 14-930-15E

Potassium Persulfate            JT Baker (VWR# JT3239-1)

(note:  this cannot be substituted with a cheaper persulfate!!)